1. Field of the Invention
The invention relates to methods of characterizing therapeutic antibodies and using information about binding specificity and more particularly as defined by a unique peptide ligand. The invention relates to therapeutic proteins which interact with alphaV containing integrin receptors.
2. Background of the Invention
Antibodies and T-cell receptor molecules possess variable regions that are responsible for specific antigenic recognition. The region of the antigen that is bound by the antibody or T-cell receptor is termed the antigenic determinant or epitope. Similarly, the variable regions of antibodies and T-cell receptors also contain determinants, or “idiotypes” that are immunogenic and are capable of initiating an anti-antibody response, an anti-idiotype (“anti-id”) immune response.
More particularly, idiotopes are associated with the variable regions of antibodies and T-cell receptors. These variable regions confer antibody and T-cell receptor specificity for antigens. Idiotypes are immune system markers on antibodies and T-cell receptors. An idiotype is immunologically defined by reactivity with more than one anti-idiotypic antibody that recognizes an idiotypic determinant or idiotope within a given idiotype; therefore, an idiotype is made up of a collection of idiotopes.
Idiotopes are best defined by their binding to monoclonal anti-idiotypic antibodies. It also should be noted that idiotopes are distinct from isotypic (immunoglobulin class-specific), xenotypic (species specific) and allo-typic (certain population sub-group specific) determinants.
Each antibody and T-cell receptor has at least one paratope that is the binding site for an antigen determinant (the epitope). A paratope may serve as an idiotope, that is, the paratope may stimulate an anti-idiotypic response in which, like the original antigen, an anti-anti-idiotopic antibody binds to an epitope within the paratope. A subset of anti-paratope anti-idiotype (“anti-id”) antibodies may mimic the immunologic properties of the original antigen and are known as “internal image” antibodies. In addition to the anti-paratopic anti-ids that mimic the original antigen, other anti-id antibodies define antibody and T-cell receptor idiotopes that also participate in the regulation of immune responses. These idiotypes are termed regulatory idiotypes and they are not necessarily “internal images” of the original antigen. For a general discussion of these background principles, see Burdette, S. and Schwartz, R., New Eng. J. of Med. 317:219 (1987).
Epitope mapping a monoclonal antibody or, in some cases, polyclonal serum, is generally understood to mean the process of deducing the exact region of the antigen or target molecule for which the antibody preparation has the highest affinity. A determinant or epitope of a target generally is understood to mean the portion of an antigen to which the most robust immune response is generated in terms of avidity and selectivity of binding. Peptides or other small fragments of an antigen can be used as immunogen (Niman et al. Proc. Natl. Acad. Sci. USA 1983, 80:4949-4951 and U.S. Pat. No. 5,030,565). In some cases peptides having unrelated sequences or structures can behave as epitopes or, mimotopes, in terms of being able to act as a binding partner for an antibody or compete for binding with the original antigen. Sequence analysis of these peptides can lead to identification of the structural and physicochemical features of the epitope. Systematic methods of altering peptide sequence and testing for antibody binding or competition between antigen and peptide have been used to perform epitope mapping. One such method, taught by Geysen et al. (Proc. Natl. Acad. Sci. USA 81: 3998-4001, 1984 and WO8403564) became widely used as it coupled the “pin” technology for solid phase peptide synthesis to ligand binding assays (sold as the Multipin Peptide Technology (PepScan) by Chiron Mimotopes, San Diego, Calif.). In numerous cases, these epitopic peptides have little or no sequence homology with the original antigenic protein or peptide (Geysen et al. Mol. Immunol. 23: 709-715, 1986). This finding lent support to the concept that, in some cases, antibodies recognized “conformational epitopes” which are only formed in three-dimensional space upon folding and twisting of the linear sequence or because of association with another peptide as in heteromultimers. Conformational epitopes are also termed mimotopes. WO8600991A1 and WO8606487A1 teach methods of determining conformational epitopes.
Another widely used method for random generation of libraries of diverse peptides for studies is the use of bacteriophage expression systems or phage display. Phage display technology provides the additional advantage that the peptide or protein displayed on the coat protein of a bacteriophage is physically linked to its genetic constituents within the phage particle (Smith, Science 228: 1315-1317,1985). Such libraries have been used as the molecular biological equivalent of the Geyson method (Devlin et al. Science 1990, 249:404-406; Pluckthun, A. Curr. Op. Biotech. 1991, 2:238-246).
Therefore, the process of epitope mapping provides information about an antigenic molecule, which when linked to information about the biological activity of the antigen or properties altered in the presence of an antibody, provide a means to deduce or understand the biological functions of the target represented by an antigen or epitope and conversely the scope of the possible effects of an antagonistic antibody which prevents normal interaction of that epitope with its naturally occurring cognate ligands.
Directed biopharmaceutic drug design is highly desirable. Therefore, an understanding of protein-protein interactions and conformation specificity in target-substrate and target-ligand interactions is important. U.S. Pat. No. 6,143,876 teaches a method of obtaining antibodies to a specific epitopes of a complex which is only revealed upon formation of that complex. WO0170984 describes in detail the significance of understanding the surface epitope bound by a Mab specific for a protein involved in coagulation as the interaction with this factor, called tissue factor, which interacts with multiple ligands sequentially.
Integrins are a family of heterodimeric transmembrane receptors that mediate cell-cell and cell-extracellular matrix adhesion. Signal transduction by integrins has been shown to regulate diversified functions, including cell differentiation, proliferation, migration, apoptosis and angiogenesis (Shimizu et al., Advances in Immunology 72: 325-385,1999; Hynes, Cell 69: 11-25,1992). Alpha-V integrins comprise a subset sharing a common alpha-V subunit combined with 1 of 5 beta subunits (beta-1, -3, -5, -6, or -8. All or most alpha-V integrins recognize the sequence RGD in a variety of ligands (vitronectin, fibronectin, osteopontin, bone sialoprotein, thrombospondin, fibrinogen, von Willebrand factor, tenascin, and agrin) and, in the case of alphaVbeta8, laminin and type IV collagen. Blocking or inhibiting the processes associated with integrin signaling is therefore a logical approach to preventing, treating or limiting the spread of cancer in the body and other diseases such as those of the eye and cardiovascular system characterized by inappropriate angiogenesis or cellular adhesion and proliferation. Accordingly, applicants have discovered a monoclonal antibody that binds and blocks the activation of alphaV containing integrin receptors, which antibody is disclosed in copending application published as WO0212501.
Given the above mentioned value that epitope mapping provides, it can be seen that the discovery of any cognate ligand for a therapeutic antibody provides not only a novel mimotope that can function as an alternate directed antigen but further provide a significant tool to measure amount and function of that therapeutic antibody. The identification of ligands to CNTO95 mAb, by identifying antagonist peptides that can inhibit binding of CNTO95 to integrins αVβ3 and αVβ5, is one strategy for the discovery of such a composition.